One interest of this laboratory has been the mechanisms of eukaryotic mRNA processing and transport. We study the HIV-1 Rev protein both as a model system and to understand the different operational circuits in the HIV life cycle. In the life cycle of HIV, the Rev protein regulates the temporal switch from the early regulatory to the late lytic phase by binding to a highly structured RRE (Rev Responsive Element) RNA. Previously, we reported on the kinetic and stoichiometric parameters of this interaction in vitro using a Surface Plasmon Resonance (SPR) biosensor. We also defined the minimal structural requirements for a functionally competent Rev. We have now analyzed the role(s) of the arginine rich RNA binding domain in RNA binding and trans-activation. The arginine rich domain could be functionally replaced by a stretch of nine arginines. Poly-arginine insertions in place of residues 24-60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for RRE. Another focus of this laboratory in recent years has been the structure-function correlation of human chemokine receptors and biochemical mechanisms of G-protein coupled receptor signaling. In particular, the structural requirements and mechanisms relating to the biological function and HIV usage by the CC and CXC chemokine receptors, CCR5 and CXCR4 were investigated. We identified a membrane proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 that constituted a bi-partite motif critical for cell surface expression. A corollary to these studies investigated the effects of naturally occurring CCR5 mutants impaired for surface expression on the function of wt receptor and its use by HIV. When molar excess of defective CCR5s were co-expressed with the wt receptor, subunit mixing was demonstrable by co-immuno-preciptation of wt and mutant CCR5s. However, over-expression of defective CCR5s (including the well characterized delta32 mutant) did not materially alter the surface presentation, chemokine mediated receptor phosphorylation or Ca++ mobilization, or usage by macrophage-tropic HIV of the co-expressed wt CCR5 protein. The ongoing studies also address the mechanistic differences underlying ligand mediated internalization and desensitization between the CC and CXC chemokine receptors. We have also identified other T cell receptors that may be linked to chemokine receptors and may be co-modulated during chemokine signaling. Among the accessory proteins encoded by HIV-1, the 27 kDal membrane associated myristoylated Nef down-modulates the surface expression of certain cellular receptors. Our studies show that Nef induced 8-30 fold and 4-8 fold down-modulation of cell surface CD4 and CCR5 respectively. Although Nef-induced corresponding decrease in the functional potential of CCR5 when measured by ligand induced receptor phosphorylation and calcium flux, there was no significant effect on the magnitude of M-tropic HIV-1 entry. In contrast to the effect on CCR5, Nef induced downregulation of CXCR4 resulted in corresponding suppression of virus entry by the T-tropic strains of HIV-1. Since the threshold levels of CCR5 for HIV entry are lower than those of CXCR4, differential effect on these receptors suggests that Nef may act as a tropism switching factor during natural infection. Besides its effect on cellular receptors, Nef also makes an important contribution to virulence. Nef has the potential to affect gene expression by interfering with cell-signaling pathways. Using cDNA microarray technology, we compared the patterns of cellular gene expression in the cell lines expressing various Nefs against the pattern in non-Nef expressing HeLa cells. We confirmed the differential expression of selected genes by RNA filter blotting. Genes expressed at higher levels included proteases, transcription factors, protein kinases, nuclear import/export proteins, adaptor molecules, and cyclins, some of which were previously implicated as being important for HIV replication and pathogenesis. The results indicate that Nef expression can alter the expression of cellular genes and suggest that this alteration in cellular gene expression may serve to optimize the cell to support the subsequent stages of viral replication.